An improved method of chromatographic analysis of protein samples is disclosed in which a Pluronic surfactant is used....http://www.google.com/patents/US20070072303?utm_source=gb-gplus-sharePatent US20070072303 - Method of chromatographic analysis of a protein solution

Method of chromatographic analysis of a protein solutionUS 20070072303 A1

Abstract

An improved method of chromatographic analysis of protein samples is disclosed in which a Pluronic surfactant is used.

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Claims(14)

1. A method for the chromatographic analysis of a protein sample comprising the step of adding a Poloxamer to a solution of the protein sample and the step of conducting a chromatographic analysis of the protein sample solution.

2. A method for the chromatographic analysis of a protein comprising preparing a diluted protein sample solution by bringing the protein concentration to a level acceptable for the chromatographic system used, adding a Poloxamer to the diluted protein sample solution and then conducting a chromatographic analysis of the diluted protein sample solution.

3. The method of claim 1, wherein the chromatographic analysis is the quantitative determination of protein content.

4. The method of claim 1, wherein the chromatographic analysis is the assessment of protein purity.

11. The method of claim 10, wherein Pluronic F68 is employed at a concentration of 100 μg/ml in ultra-pure water in the protein sample solution.

12. The method of claim 10 wherein, Pluronic F68 is employed at a concentration of 0.1% in sodium acetate buffer at pH 3.8 in the protein sample solution.

13. A method for the chromatographic analysis of the purity and/or quantity of a protein in a sample, comprising chromatographically analyzing the protein in a sample that contains a Poloxamer.

14. The method of claim 13, further comprising data manipulation to determine purity and/or quantity of the protein.

Description

FIELD OF THE INVENTION

This invention relates to methods for the analysis of proteins.

More specifically, it relates to the analysis of proteins by chromatographic methods (e.g. HPLC). By “analysis” of proteins is meant here both the quantitative determination and the assessment of purity of a protein.

BACKGROUND OF THE INVENTION

Proteins in pharmaceutical products must be analysed to quantify the protein and to ensure purity. This permits correct and reproducible dosing to the patient.

Analytical techniques are necessary to quantify the pharmaceutical protein as well as impurities, aggregates, and degradation products. In the case of multimeric proteins, such as dimers, analytical techniques are required to detect the presence and extent (i.e. quantity) of dissociation. Such analytical techniques should be precise (i.e. the degree of variation when the same sample is tested multiple times should be low), and accurate (i.e. the measured value should be as close as possible to the actual value). Reproducibility is also important.

An example of an analytical assay that may be used with proteins is size exclusion chromatography (SEC), in which a protein in aqueous solution is passed over a solid or gel phase that separates mixtures of protein by differences in their molecular weight. The resulting chromatogram shows one or more peaks associated with the protein(s) in a sample, which may be identified by molecular weight. The area under a peak associated with a given protein can be used to quantify the amount of that protein in the sample. The shape of the peak may be used to assess purity.

Another example of an analytical assay that may be used with proteins is high performance liquid chromatography (HPLC), in particular reverse phase high performance liquid chromatography (RP-HPLC). A sample containing the pharmaceutical protein is passed through a column which separates the protein from any impurities. The protein and any impurities elute as peaks on a chromatogram. As with SEC, the area under a peak associated with a given protein can be used to quantify the amount of that protein in the sample. The shape of the peak may be used to assess purity.

In chromatographic methods, such as those mentioned above, the protein must be dissolved in an aqueous solvent and diluted to an extent acceptable for the chromatographic system used. During handling of the aqueous protein solution, the risk exists that part of the protein is lost by adsorption to handling and containment equipment, such as glass or plastic walls of capillaries, test-tubes, beakers, syringes, etc., making quantitation of protein difficult. The adsorption of protein leads to variations in the results that detract from the assay precision, accuracy and reproducibility

Surfactants have been used in the purification of proteins by SEC, and in assays in which the molecular weight of a new protein is determined, see for example EP 0 530 937 and DE 39 17 949.

It would be desirable to have a chromatographic method of protein analysis for quantifying protein and/or assessing purity, in which assay precision, accuracy and reproducibility are improved by avoiding variations due to protein adsorption.

SUMMARY OF THE INVENTION

It has now been discovered that a surfactant of the class of Poloxamers avoids protein loss and at the same time does not interfere with the chromatographic analysis.

In a first aspect, the invention provides, in a method of chromatographic analysis of a protein sample solution, the improvement consisting in adding a Poloxamer to the protein sample solution.

In a second aspect, the invention provides, in a method of chromatographic analysis of a protein including the step of preparing a diluted sample for bringing the protein concentration to a level acceptable for the chromatographic system used, the improvement consisting in adding a Poloxamer to the diluted sample solution.

In a third aspect, the invention provides a method for the chromatographic analysis of the purity and/or quantity of a protein in a sample, the method comprising a step of preparing the sample to contain a Poloxamer.

DETAILED DESCRIPTION OF THE INVENTION

The inventors have found that by including a Poloxamer in a protein solution to be assayed for purity and protein content, protein adsorption is decreased, leading to increased assay precision, accuracy and reproducibility.

In the context of the present application, the term “analysis” is meant to encompass an analytical process whereby the purity of and/or quantity (e.g. concentration) of a protein in a sample is determined, preferably the quantity. In a preferred embodiment, the method of the invention comprises a method for the analysis of the purity and/or quantity of a protein in a sample, the method comprising a step of preparing the protein sample to contain a Poloxamer, and performing a step of chromatography, preferably a step of SEC or RP-HPLC. Preferably the step of chromatography is followed by a step of data manipulation to determine purity and/or quantity of the protein. The quantity of protein may be determined using data from calibration with a standard. Calibration may be carried out before or after analysis of the sample.

Poloxamers are block copolymers made of poly(oxyethylene)-poly(oxypropylene) blocks with M.W. ranges from 1,000 to >16,000. Their main characteristic is that the poly(oxyethylene) segments are hydrophobic and the poly(oxypropylene) segments hydrophilic. These substances behave as non-ionic surfactants and are generally known with the commercial name “Pluronics”.

Many grades of Pluronics at various Molecular Weight and concentration ranges can be used in accordance with this invention.

Other polymers having properties similar to those listed above may also be used in the methods of the invention. The preferred surfactant is Pluronic F68 (Poloxamer 188), and surfactants having similar properties.

Therefore, this invention relates to an improved method of chromatographic analysis of a protein including the step of preparation of a protein sample with a protein concentration acceptable for the chromatographic system used, the improvement consisting in adding a Poloxamer, preferably Pluronic F68, to the protein sample solution.

In the Examples which follow, Pluronic F68 (Poloxamer 188) is used at the concentration of 100 μg/ml in ultra-pure water. It is however understood that the use of different grades of Pluronic and different concentrations of the same is encompassed by the present invention.

EXAMPLE1 Improved Sample Preparation in the SEC Method of Quantitative Determination of FSH

The purpose of this study was to qualify an improved sample preparation in the Size Exclusion Chromatography (SEC) method for protein content in a preparation containing rec-FSH (Fertinex, in this case Fertinex 75 IU).

The modification implemented was the use of a solution including 100 μg/ml Pluronic F68 in ultra-pure water for preparation of all protein solutions (sample, control sample and standard) in order to control losses of protein due to adsorption.

Samples consisting of mixtures of heterodimeric FSH (FSH is composed of an α-subunit and a β-subunit) and dissociated FSH were prepared and analysed. The method allowed the quantitation of heterodimeric FSH and free subunits.

This study shows that a single point calibration curve using drug product reference standard is suitable to determine protein content with a good total precision of 2.0% and that the method is linear within the range tested (26.6 to 160 μg/ml).

In addition, both Waters and Varian HPLC systems can be used as shown during the study. The difference in the mean protein content results of all batches is lower than the total precision of the method.

It is important to emphasize that this change did not have any impact on the original SEC method itself.

More specifically, the modification of the sample and standard preparations were as follows:

Use of a 100 μg/ml Pluronic F68 in ultra-pure water for the preparation of the samples and reference standard.

Blank solution used through the analytical sequence is the 100 μg/ml Pluronic F68 solution.

The preparation for SST control sample as well as control samples injected over the analytical session are performed by reconstituting and pooling enough ampoules to allow the injections of the system suitability as well as for the controls which are injected throughout the analytical session.
Materials and Equipment
Materials

The column was conditioned for an hour with the mobile phase (phosphate buffer 0.1 M; Na2SO4 0.1 M, pH 6.70), at a flow rate of 0.70 ml/minute. The column was maintained at approximately 4° C. throughout the analysis.

Sample solutions: the sample solutions were prepared to have varying amounts of heterodimeric FSH (Fertinex) and dissociated FSH, in a solution containing Pluronic F68 (100 μg/ml).

Control solutions: control solution was prepared using FSH dissolved in a solution containing Pluronic F68 (100 μg/ml).

The standard and sample solutions were injected (100 μl), and the column was eluted at a constant flow rate of 0.70 ml/min. Detection was by UV absorption at 214 nm. Area under the peaks was used to determine heterodimeric FSH and the respective FSH subunits.

Results

The precision of an analytical method expresses the closeness of agreement (degree of scatter) between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions. Precision may be considered at three levels: repeatability (or intra-assay precision), intermediate precision and reproducibility. During this study, repeatability, intermediate precision as well as reproducibility of the assay were addressed.

In five independent analytical sessions, three Fertinex drug products batches were quantified against the standard (calibration curve).

With the results of Fertinex 75 IU presented in Table 1, an analysis of variance (ANOVA) Nested design was used to estimate the repeatability (or intra-assay precision), intermediate precision and reproducibility (total precision) of the analytical method. The total number of results under study was 45. The results obtained were as follows:

Repeatability (or intra-assay precision): 1.3%

Intermediate precision: 1.0%

Reproducibility (total precision): 1.6%

Overall results are tabulated in the following Table 1:

TABLE 1

Protein content results (μg/ampoule)

obtained with calibration curve standard

Batch number

Run 1

Run 2

Run 3

Run 4

Run 5

17301010

6.41

6.42

6.32

6.56

6.41

17315040

6.13

6.06

5.94

6.11

6.15

17318040

5.54

5.58

5.49

5.59

5.51

Control sample

6.00

6.18

6.04

6.21

6.07

The standard results obtained during the precision and ruggedness studied were used to address the linearity and range of the analytical method. The approach for the statistical analysis was to check variance homogeneity (Cochran C and Bartlett tests), lack of fit and perform regression analysis (correlation coefficient). Run 1 to Run 5 were performed with a Waters systems whereas Run 6 to Run 8 were performed with a Varian system.

TABLE 2

Statistical results for linearity

Run 1

Run 2

Run 3

Run 4

Run 5

Run 6

Run 7

Run 8

Cochran's C test

0.99

0.98

0.99

0.99

0.99

0.98

0.96

0.98

Bartlett's test

0.99

0.98

0.99

0.99

0.99

0.98

0.96

0.98

Lack of fit

0.97

0.18

0.83

0.92

0.81

0.89

0.03

0.07

Slope

1.0058

0.9926

1.0028

1.0066

0.9940

0.9903

1.0202

0.9888

Intercept

6.1557

6.2238

6.1574

6.2099

6.2249

5.5055

5.7437

5.5214

Correlation coeff.

1.0000

0.9999

0.9999

0.9999

1.0000

0.9999

0.9998

0.9999

As can be seen, the p-values of Cochran C and Bartlett tests are always greater than 0.05 which means that there is no statistically significant difference amongst the standard deviations at 95% confidence level and a correlation coefficient higher than 0.9980 is always met.

Lack of fit to determine whether linear regression is an adequate model to describe the observed data was performed. Based on this statistical test, linear regression resulted to be the best model to describe the data even if the p-value of Run 7 is not greater than 0.05. For that particular analytical session, the linear regression is still the best model with a 99.96% fit. Other models, such as square root and exponental models were tested and did not show a better fit of the data observed.

In a spiking experiment, known amounts of either heterodimeric FSH or dissociated FSH were added to a sample of FSH (“spiking”). The resulting peaks for heterodimeric and/or dissociated subunits were evaluated for % recovery and for % purity.

The modification implemented in the analytical method is the use of a solution including 100 μg/ml Pluronic F68 in ultra-pure water for preparation of all protein solutions (sample, spike solution) to control losses of protein due to adsorption. The modifications were made specifically to increase the precision of the method, without impacting the chromatography of the method, to enable a more precise and accurate purity determination.

In the frame of this study, determination of the precision of the method was also investigated. The precision of the method (Total precision 1.8%) was slightly improved when compared to the precision of the method observed during validation of the analytical method without introduction of Pluronic F68 (Precision 2%). Routine recovery of the spike solutions at 100% indicates a good accuracy.

An additional spiking experiment was performed to further determine accuracy of the method. It was observed that different area under the peak is obtained for the spiking solution with and without Pluronic F68. The area of the spiking solution with Pluronic F68 in the sample preparation is approximately two times greater than the spiking solution prepared without the use of Pluronic F68.

It is believed that the different areas observed are due to adsorption of the dissociated sub-units on the polypropylene material used for sample preparation.

It can be seen in the following Tables 3 and 4 that the recovery of spiked free sub-units of FSH is within the range 95%-105%. In addition it can also be seen that the precision [reported as coefficient of variability (CV %)] over the five runs are ranging from 1.0% to 1.5% for both purity and recovery of spike. The lower the CV %, the lesser the variability between runs.

This is a substantial improvement over samples prepared without Pluronic F68.

TABLE 3

% Recovery of spike results

Batch#

Run 1

Run 2

Run 3

Run 4

Run 5

Mean

CV %

17343109

98.0%

98.1%

100.3%

99.6%

99.4%

99.1%

1.0%

17349129

98.9%

99.6%

102.1%

100.7%

102.2%

100.7%

1.5%

17301010

99.8%

100.7%

102.0%

99.2%

99.8%

100.3%

1.1%

17304010

99.8%

102.4%

101.6%

101.0%

99.9%

100.9%

1.1%

TABLE 4

% Purity of spike results

Batch#

Run 1

Run 2

Run 3

Run 4

Run 5

Mean

CV %

17343109

97.0%

94.4%

93.9%

96.3%

96.3%

95.6%

1.4%

17349129

96.2%

93.4%

93.3%

95.5%

94.4%

94.6%

1.4%

17301010

96.8%

95.0%

94.1%

96.9%

97.2%

96.0%

1.4%

17304010

95.9%

93.6%

94.0%

95.9%

97.0%

95.3%

1.5%

In the frame of another study protocol, the analysis of drug product batches with and without the use of Pluronic F68 was performed. One of the results is that the area under the peak of the spiking solution (dissociated r-hFSH) is approximately multiplied by two with the introduction of Pluronic F68. This phenomenon is most probably due to adsorption of free sub-units on the material used during sample preparation if Pluronic F68 is not present. To determine if this increase of area has an impact on the analysis, a spiking experiment at three levels without and with Pluronic F68 was performed. The following samples were tested in duplicate:

1. Sample Preparation with Pluronic F68

Sample without spiking solution;

Sample spiked with dissociated r-hFSH (2 μg by injection);

Spiking solution (2 μg dissociated r-hFSH by injection);

Sample spiked with dissociated r-hFSH (1.5 μg by injection);

Spiking solution (1.5 μg dissociated r-hFSH by injection);

Sample spiked with dissociated r-hFSH (1 μg by injection);

Spiking solution (1 μg dissociated r-FSH by injection).

The results are presented in table 5:

TABLE 5

Results of sample preparations using

Pluronic F68: area under peak

%

%

Area of

Recovery

%

Recovery

Spiking level

spiking solution

of areas

Purity

of spike

100% (2 μg/inj)

2037915

N/A

95%

101%

75% (1.5 μg/inj)

1545486

76%

94%

101%

50% (1 μg/inj)

1001055

49%

94%

102%

From the above table, it can be seen that the addition of Pluronic F68 gives good results in term of % recovery of spike, being close to the theoretical recovery (100%) and % Recovery of areas. This latter parameter is calculated by dividing the area of the spiking solution at the defined spiking level by the area of the spiking solution at 100%. The result (i.e. 76% for spiking with 1.5 μg/injection) is compared with the theoretical spiking level performed (i.e. 75%).
2. Sample Preparation Without Pluronic F68

The following samples were tested in duplicate:

Sample without spiking solution;

Sample spiked with dissociated r-hFSH (2 μg by injection);

Spiking solution (2 μg dissociated r-hFSH by injection);

Sample spiked with dissociated r-hFSH (3 μg by injection);

Spiking solution (3 μg dissociated r-hFSH by injection);

Sample spiked with dissociated r-hFSH (4 μg by injection);

Spiking solution (4 μg dissociated r-hFSH by injection)

Results are shown in the following Table 6:

TABLE 6

Results of sample preparations without

Pluronic F68: area under peak

%

Area of

Recovery

%

%

Spiking level

spiking solution

of areas

Purity

Recovery

100% (2 μg/inj)

922106

N/A

90%

105%

150% (3 μg/inj)

1744753

189%

89%

104%

200% (4 μg/inj)

2702520

292%

89%

104%

For the results obtained without the use of Pluronic F68 in the sample preparation, the recovery of the spike solution deviates 4-5% from the theoretical recovery (100%). For % recovery of areas, the results deviate significantly from the theoretical spiking level performed, due to a too low area of the spike solution.

It is believed that the different results obtained with and without Pluronic F68 can be explained by the adsorption of the test samples on the material used during sample preparation if Pluronic is not included in the assay solutions. The area under the peak of spiking solution obtained for the same spiking level (100%) is approximately two times greater when Pluronic F68 is present and avoids the adsorption.

The adsorption of free sub-units can be calculated by the difference between the area under the peak of the spiking solution with Pluronic F68 and the area of the spiking solution without Pluronic F68 at 2 μg per injection (100%) spiking level [i.e. subtracting the area under the peak at a spiking level of 100% without Puronic F68 (Table 6: 922106) from the area under the peak at a spiking level of 100% with Pluronic F68 (Table 5: 2037915)]. The difference corresponds to an area of 1′115′809. This area reflects the amount of dissociated sub-units adsorbed during sample preparation when Pluronic F68 is not present, and can be used to correct the area obtained in absence of Pluronic F68. The recoveries of area calculated taking into account this correction can be seen in table 7. The data are closer to the theoretical spiking level.

TABLE 7

% Recovery of area with and without taking into account

adsorption (in the absence of Pluronic F68)

% Recovery of areas taking into account adsorption

Spiking level

No

Yes

3 μg/inj (150%)

189%

140%

4 μg/inj (200%)

292%

187%

The purity results presented in tables 5 and 6 differ without and with the introduction of Pluronic. This difference is also believed to be an effect of adsorption phenomenon. Percent purity is significantly more accurate when Pluronic F68 is used.

Several product batches were tested by SEC for purity without and with introduction of Pluronic F68 in sample preparation. The results can be seen in table 8:

TABLE 8

% Recovery and % Purity results of drug product

tested without and with Pluronic F68

Without Pluronic

With Pluronic

Batch#

% Recovery

% Purity

% Recovery

% Purity

17353066

98%

89%

96%

94%

17303019

101%

91%

100%

97%

17306019

104%

91%

97%

98%

17309029

100%

90%

100%

97%

17323049

101%

92%

98%

97%

17324049

98%

94%

97%

99%

17340089

100%

93%

99%

97%

17341089

101%

91%

98%

98%

17345119

100%

90%

100%

96%

17305019

105%

88%

99%

101%

17315039

99%

91%

101%

95%

17302019

100%

87%

99%

96%

17501019

106%

86%

99%

96%

Mean

101.0%

90.2%

98.7%

97.0%

Standard

2.5%

2.3%

1.4%

1.8%

deviation

CV %

2.5%

2.5%

1.5%

1.8%

Purity and Recovery of Spiking Solution

Based on the data presented in Table 8 above, it can be seen that the difference between the recovery without and with introduction of Pluronic is about 2%. This difference is statistically significant when an ANOVA at 95% confidence level is performed (p-value 0.008). In addition, when looking at the same table one can also see a statistically significant difference of approximately 7% in purity with and without introduction of Pluronic F68 (ANOVA p-value 1.2 10−8).

Furthermore, it can be seen from Table 8 that the CV % is lower when Pluronic F68 was included in the sample solution [2.5% without Pluronic F68 VS 1.5 and 1.8% with Pluronic F68]. A lower CV % indicates a lesser degree of variability between runs.

These statistical differences in purity and recovery of spiking solution can be explained by adsorption phenomenon which occurs during sample preparation when Pluronic F68 is not used (see the following Table 9).

In Table 9 and subsequent Tables, “Dimers and Aggregates” refers to aggregated FSH molecules and dimers of heterodimeric FSH that are generally considered to be undesirable.

TABLE 9

Area variability: area under peaks of heterodimeric FSH, free subunits and dimers and aggregates

Dimers and aggregates

Total area of FSH spiking solution

Total area of unspiked sample

area of sample

Batch #

Area

CV %

Area

CV %

Area

CV %

Pluronic

No

Yes

No

Yes

No

Yes

No

Yes

No

Yes

No

Yes

17353066

1409832

2658545

2.2

1.7

7377193

7767243

0.4

2.5

85841

90638

10.33

3.92

17303019

1438014

2513326

0.5

0.2

6136762

6312492

4.8

2.2

65913

64896

2.28

3.05

17306019

1438014

2513326

0.5

0.2

6029360

6474462

1.2

1.2

69387

65633

3.06

6.49

17309029

1121632

2380100

3.1

2.4

6554845

6906608

0.5

0.5

74694

83950

4.36

5.06

17323049

1300382

2429203

1.2

1.5

5692277

6157225

1.8

1.5

64801

67053

5.47

7.62

17324049

1300382

2429203

1.2

1.5

5856669

6129612

4.6

1.5

61088

58535

5.49

0.72

17340089

1449322

2564021

2.3

3.4

6316392

6577138

1.7

0.7

55831

64916

1.93

3.53

17341089

1449322

2564021

2.3

3.4

6311904

6658876

1.3

0.4

51410

66879

6.79

2.36

TABLE 9

Area variability: area under peaks of heterodimeric FSH, free subunits and dimers and aggregates

Dimers and aggregates

Total area of FSH spiking solution

Total area of unspiked sample

area of sample

Batch #

Area

CV %

Area

CV %

Area

CV %

Pluronic

No

Yes

No

Yes

No

Yes

No

Yes

No

Yes

No

Yes

17345119

1121632

2380100

3.1

2.4

5678718

6094436

0.5

0.3

61319

71681

5.86

0.98

17305019

1416915

2609854

3.5

0.8

6105716

6366397

3.4

1.3

81412

76271

8.77

1.96

17315039

1207140

2528909

7.4

5.7

6629111

7129130

1.4

0.6

69514

65511

1.74

3.77

17302019

1207140

2528909

7.4

5.7

6306795

6975041

3.8

0.6

97979

94241

5.28

8.59

17501019

1416915

2609854

3.5

0.8

7416153

7580193

1.1

2.1

69441

67857

6.4

6.1

Mean

1328972

2516105

2.9

2.3

6339377

6702219

2.0

1.2

69895

72159

5.2

4.2

CV %

9.5%

3.5%

76%

81%

8.7%

8.1%

77%

63%

18%

15%

50%

59%

Dissociated FSH area of spiked sample

Total area of spiked sample

Batch #

Area

CV %

Area

CV %

Pluronic

No

Yes

No

Yes

No

Yes

No

Yes

17353066

2132665

3052448

2.5

1.08

8618890

9989197

0.92

0.41

17303019

1908454

2640090

4.5

0.43

7634720

8866597

1.52

0.83

17306019

1920007

2564678

1.2

0.81

7739564

8723677

1.12

0.58

17309029

1697129

2520633

6.0

2.01

7695502

9270545

2.16

1.48

17323049

1697821

2542802

1.8

1.14

7033218

8410312

1.14

1.09

17324049

1596022

2439971

6.4

2.61

7022864

8324957

2.32

0.81

17340089

1862023

2678583

3.2

1.27

7763745

9078480

0.79

1.49

Dissociated FSH area of spiked sample

Total area of spiked sample

Batch #

Area

CV %

Area

CV %

Pluronic

No

Yes

No

Yes

No

Yes

No

Yes

17341089

1977742

2642383

1.3

1.16

7846421

9034549

0.31

0.27

17345119

1616907

2555312

5.7

1.80

6808816

8480285

1.21

1.17

17305019

2044400

2479940

1.2

2.99

7863722

8851837

3.27

0.95

17315039

1722445

2785597

5.0

0.08

7721964

9750217

2.01

0.41

17302019

1943343

2681371

11.0

2.72

7543478

9363087

2.68

2.07

17501019

2354166

2847934

0.4

0.5

9364548

10138501

0.2

0.35

Mean

1882548

2648596

3.9

1.4

7742881

9098634

1.5

0.9

CV %

12%

6.3%

77%

65%

8.6%

6.4%

61%

59%

It can be seen from Table 9 that the precision (CV %) of the areas used for the purity and recovery of spiking solutions calculation is almost always better with Pluronic F68 than without the introduction of Pluronic F68 [lower CV % indicates improved precision].

The adsorption rate of dimers and aggregates, free sub-units and monomer are different as one can see when calculating the ratio mean area with Pluronic F68/mean area without Pluronic F68. As can be seen in table 10, the % Area increase with Pluronic F68 is not constant depending on the area considered.

TABLE 10

% Area increase with introduction of Pluronic F68

% Area

Area considered

increase

Total area of spiking solution

6%

Total area of unspiked sample

89%

Dimers and aggregates areas of unspiked sample

41%

Dissociated sub-units area of spiked sample

3%

Total area of spiked sample

18%

To determine purity, spiking of free sub-units must be performed due to the fact that the free sub-units peak is not resolved from main peak during testing of sample preparation. The % Purity (or % Monomer) is expressed by the formula below:
%Purity=A+B-C-DA
Where: A: Total area of sample without spike

B: Total area of spiking solution

C: Sub-units peak area of sample spiked

D: Dimers and aggregates in sample without spike

The acceptance criteria for recovery of spike is expressed as follows:
%Recovery=EA+B
Where: A: Total area of sample without spike

B: Total area of spiking solution

E: Total area of spiked sample

Taking the above formulas into consideration, calculation of purity with and without introduction of Pluronic F68 can be performed. In addition, to take into consideration the effect of Pluronic F68, calculations can also be done with areas without introduction of Pluronic F68 taking into account the adsorption effect. The formulas will be the following:
%Puritycorrected=A⨯Ra+B⨯Rb-C⨯Rc-D⨯RdA⨯Ra%Recoverycorrected=E⨯ReA⨯Ra+B⨯Rb
Where: A: Total area of sample without spike

B: Total area of spiking solution

C: Sub-units peak area of sample spiked

D: Dimers and aggregates in sample without spike

E: Total area of spiked sample

Rx: Ratio mean area X with Pluronic F68 divided by mean area X without Pluronic F68.

EXAMPLE 3 Interferon Beta-1a Assay by RP-HPLC: Qualification of a One Standard Point Approach Versus a Standard Curve Approach

This Example shows how, in the case of Interferon beta-1a assay by RP-HPLC, it was possible to modify a standard curve approach, hereinafter referred to as “current assay”, to a One Standard Point approach by applying the improved method of this invention, that is, by using a Pluronic surfactant to avoid sample losses.

In the drug substance sample preparation for the improved method of the invention, Interferon beta-1a was diluted 1 to 7 using as the dilution buffer 0.05M sodium acetate containing 0.1% Poloxamer 188 (Pluronic F68) at pH 3.8 instead of 0.05M sodium acetate at pH 3.8 without Poloxamer.

The optimized RP-HPLC assay was qualified according to the ICH Guidelines and the following characteristics were addressed:

Range of linearity: The linearity was verified for the injection of Interferon-beta-1a in the range of 3.3 to 8.8 μg, with an intercept statistically not different from zero. These results support the One Standard Point approach.

Precision: The precision of the optimized assay using the Drug Product Standard and the One Standard Point approach was compared to the precision obtained with the current assay using the Drug Substance Standard and the Standard curve approach. The overall precision obtained for one replicate with the optimized assay using Pluronic F68 (pooled CV of 1.2% for all Drug Substances and Drug Products samples) was shown to be better than the precision of 1.9% obtained for one replicate with the current assay, not using Pluronic F68.

Specificity: the components that can be observed in the optimized assay are already present in the Drug Substance and Drug Product formulations analyzed in the current assay which was validated for specificity. The presence of Poloxamer 188 in the new Standard was demonstrated to not interfere in the optimized assay. As no additional components are introduced in the optimized assay, the optimized assay is therefore specific.

Accuracy: The accuracy of the optimized assay was assessed by comparison of the results obtained with the optimized assay versus the results obtained with current assay, these last results being considered as the reference value. For this purpose, several Drug Substances and Drug Products (Liquid formulation containing HSA, Liquid formulation HSA free) were analyzed. In all cases, the ratio Optimized/Current Interferon-beta-1a assay was not statistically different from 100%, which demonstrates that the two Interferon beta-1a assays generate statistically equivalent results.

In conclusion, the method has a better precision than the current assay, and generates accurate results statistically equivalent to those obtained with the is current assay.

This invention has been described with reference to examples of both quantitative determination of proteins and purity assessment of the same in both SEC and RP-HPLC chromatographic methods.

It will be apparent that many other equivalent examples can be done without departing from the spirit and scope of the invention that is defined by the following claims.